Project Summary Chronic infections with the protozoan parasite and causative agent of human Chagas disease, Trypanosoma cruzi, are notoriously challenging to treat. The available drugs often fail to achieve sterilizing cure under the current treatment regimens. Ergosterol biosynthesis inhibitors (EBIs) also failed to clear parasites from chronic patients in recent clinical trials. These findings, coupled with results from animal models showing selective survival of T. cruzi in certain tissues after EBI treatment, raise the possibility that characteristics of the local tissue environment impact susceptibility of intracellular parasites to trypanocidal drugs. In the cancer field, it is well-established that differences in the metabolic state of tumor cells, due to heterogeneity of cell populations and their environments, can lead to `metabolic resistance' and treatment failure. Because T. cruzi colonizes diverse tissues in the mammalian host, each with its own unique metabolic signature, we hypothesize that comparable mechanisms of metabolic resistance to anti-trypanosomal drugs may contribute to failure to achieve parasitological cure in chronic T. cruzi infection. Consistent with this idea, preliminary studies show that a single change in the composition of the cell culture medium protects intracellular T. cruzi amastigotes from the lethal effects of EBIs. The goals of this proposed study are to determine the role of glutamine metabolism in sensitizing T. cruzi amastigotes to EBIs using a combination of molecular genetic and biochemical approaches (Aim1) and to determine the broader impact of metabolic environment on the efficacy of the next generation of candidate anti-trypanosomals by re-screening the ChagasBox collection of T. cruzi growth inhibitors under diverse conditions (Aim 2). As a case study for metabolic resistance, the proposed study addresses a critical gap in our understanding of how diverse cellular and metabolic environments, such as those encountered by T. cruzi in vivo, impact the ability to effectively eliminate this pathogen from infected hosts, a key current challenge in the treatment of Chagas disease.